The overarching goal of this project is to develop a platform for the chemoenzymatic synthesis and evaluation of trehalose analogs as tools for investigating trehalose metabolism in mycobacteria. Trehalose is a non-mammalian disaccharide that is essential for cell-wall biosynthesis and function in Mycobacterium tuberculosis (Mtb), the bacterium that causes human tuberculosis (TB). Because trehalose is integral to Mtb pathogenesis but absent from the human host, it is an attractive target for diagnostic and therapeutic development. Chemically-modified trehalose analogs have begun to emerge as important tools in TB research-for instance, detectable trehalose analogs have recently been explored as mycobacteria-specific imaging probes. However, a major hurdle to progress in this area is the difficulty associated with synthesizing trehalose analogs using conventional approaches. A central goal of this proposal is to develop a chemoenzymatic method for the rapid and efficient preparation of trehalose analogs employing the heat-stable enzyme TreT from Thermoproteus tenax, which is the focus of Aim 1. Using TreT, we show that a broad range of novel trehalose analogs can be prepared quickly (= 60 min) in high yield (up to > 99%) in a single step from readily available glucose analogs. Furthermore, the aqueous reaction conditions enable direct transfer of reaction mixtures to mycobacterial cells to allow rapid administration and evaluation of trehalose-based probes or inhibitors. In Aim 1, we intend to further develop and characterize the TreT method, with a focus on expanding reaction scope and scale. We will also generate a panel of trehalose analogs for investigating two processes that are important for mycobacterial pathogenesis: trehalose recycling and biofilm formation. Aim 2 involves using trehalose analogs to evaluate the substrate specificity of the trehalose-recycling transporter SugABC-LpqY, which will inform our efforts to design trehalose-based probes or inhibitors that hijack SugABC-LpqY to enter the cell. In Aim 3, we will evaluate trehalose analogs as inhibitors of mycobacterial biofilm formation, which may contribute to Mtb persistence and drug tolerance during infection. In concert, we will also test a panel of trehalose metabolism mutants for impaired biofilm formation to explore the genetic link between trehalose and mycobacterial biofilms.